Immunoassays, such as Western blots and Enzyme Linked Immunosorbent Assays (ELISAs), have long been the primary method for target protein detection and quantification in biological samples, including transgenic crops. ELISAs have remained a popular choice because of their throughput, sensitivity, and selectivity. The drawback to ELISAs and other immunoassays is the need for high quality antibodies that may not be readily available and the significant time (approximately 10-20 months) and resources needed to develop an assay. Additionally, a separate assay is typically needed for each protein of interest. Multiplexed methods are desirable as genetically engineered crops featuring stacked traits/proteins become more prevalent.
Mass spectrometry has become an important tool for targeted protein detection and quantification. The first fully validated liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of multiple target proteins in transgenic maize was recently reported. In that study, transgenic sample extracts, along with calibration curves made by spiking recombinant proteins into null (non-transgenic) tissue extract, were efficiently digested with the protease trypsin. Signature peptides were detected and quantified by multiple reaction monitoring (MRM) as protein surrogates. In a similar manner, liquid chromatography coupled with multiple reaction monitoring (LC-MRM) was used to quantify a Bacillus thuringiensis (Bt) protein, Cry1Ab, in transgenic maize leaves. In both cases, protein reference standards were required. Therefore, methods are needed for detecting and quantifying polypeptides in a plant without the use of a reference standard.